This study investigates the impact of cytosine methylation on the DNA binding specificities of human transcription factors (TFs). Using high-throughput systematic evolution of ligands by exponential enrichment (HT-SELEX), researchers analyzed 542 human TFs and found that many prefer CpG-methylated sequences, particularly those in the extended homeodomain family. Structural analysis revealed that homeodomain TFs recognize methylcytosine through direct hydrophobic interactions with the methyl group. The study shows that DNA methylation significantly affects TF binding, with some TFs showing increased or decreased binding to methylated or unmethylated sequences. The results indicate that many developmentally important TFs prefer mCpG-containing sequences, and that methylation can influence TF binding in both in vitro and in vivo settings. The study also highlights the structural basis of mCpG preference, showing that homeodomains recognize methylcytosine through hydrophobic interactions. The findings contribute to understanding the role of DNA methylation in epigenetic inheritance and transcriptional regulation. The study provides a comprehensive analysis of TF binding specificities and their relationship with DNA methylation, revealing that many TFs are sensitive to methylation, and that methylation can influence TF binding in both positive and negative ways. The results have implications for understanding the regulation of gene expression and the role of DNA methylation in cellular differentiation and reprogramming.This study investigates the impact of cytosine methylation on the DNA binding specificities of human transcription factors (TFs). Using high-throughput systematic evolution of ligands by exponential enrichment (HT-SELEX), researchers analyzed 542 human TFs and found that many prefer CpG-methylated sequences, particularly those in the extended homeodomain family. Structural analysis revealed that homeodomain TFs recognize methylcytosine through direct hydrophobic interactions with the methyl group. The study shows that DNA methylation significantly affects TF binding, with some TFs showing increased or decreased binding to methylated or unmethylated sequences. The results indicate that many developmentally important TFs prefer mCpG-containing sequences, and that methylation can influence TF binding in both in vitro and in vivo settings. The study also highlights the structural basis of mCpG preference, showing that homeodomains recognize methylcytosine through hydrophobic interactions. The findings contribute to understanding the role of DNA methylation in epigenetic inheritance and transcriptional regulation. The study provides a comprehensive analysis of TF binding specificities and their relationship with DNA methylation, revealing that many TFs are sensitive to methylation, and that methylation can influence TF binding in both positive and negative ways. The results have implications for understanding the regulation of gene expression and the role of DNA methylation in cellular differentiation and reprogramming.